Neuropathy remedies

ABSTRACT

The present invention provides a neurodegenerative disease therapeutic agent containing as its active ingredient a (15R)-isocarbacycline derivative indicated by the following formula [I] or a 15-deoxy-isocarbacycline derivative indicated by the following formula [III]: 
                 
 
(wherein, R 1  represents a C 1 -C 6  alkylene group, and R 2  represents a hydrogen atom, a C 1 -C 7 , alkyl group or protective group); or, 
                 
 
(wherein, R 1  and R 2  are the same as those defined in formula [I]).

TECHNICAL FIELD

The present invention relates to a neurodegenerative disease therapeuticagent, to a therapeutic agent for degenerative diseases presentingdementia symptoms and, particularly, to an Alzheimer's diseasetherapeutic agent. More particularly, the present invention relates to aclinically applicable neurodegenerative disease therapeutic agent, to atherapeutic agent for degenerative diseases that present dementiasymptoms and, in particular, to an Alzheimer's disease therapeuticagent, that is highly effective in improving learning and memorydisorders and has minimal adverse side effects such as toxicity andblood pressure reduction.

BACKGROUND ART

Neurodegenerative disease is the general term for a group of diseases ofunknown cause resulting in neural disorders at a specific site. Morespecifically, examples of degenerative diseases of the cerebrum includeAlzheimer's disease, and Pick's disease, examples of degenerativediseases of the cerebral basal ganglia include Parkinson's disease andHuntington's disease, examples of degenerative diseases of thecerebellum include spinocerebellar atrophy, and degenerative diseases ofthe spinal cord include amyotrophic lateral sclerosis.

Since the cause of these neurodegenerative diseases is unknown, it isdifficult to treat them with etiogenic therapy, making it necessary torely upon nosotropic therapy.

For example, although all of the drugs currently approved for use astherapeutic agents of Alzheimer's disease are acetylcholine nervoussystem activators, they are nosotropic therapeutic agents developed onthe basis of the pathological finding that the acetylcholine nervoussystem is significantly impaired in Alzheimer's disease patients. Inaddition, in actual Alzheimer's dementia, it has been demonstrated thatthe acetylcholine nervous system is not the only system that isimpaired, and with respect to this point as well, there thought to belimitations on the effects of acetylcholine nervous system activators.

However, due to the recent progress in disease research at the molecularlevel, it has been demonstrated that many neurodegenerative diseasesshare a common characteristic in that neuropathy/cell death is inducedby the polymerization and accumulation within the cell of abnormalproteins unique to each disease.

For example, in the brain of an Alzheimer's disease patient,amyloid-like extracellular deposits referred to as senile plaque, andfibrous compounds composed mainly of phosphorylated tau protein(neurofibrillary tangle), are observed in parallel with pathologicalcondition. The major component of senile plaque is an insoluble proteinadopting a β sheet structure composed of 40 to 43 amino acid residuesreferred to as amyloid β protein (Aβ). This protein has beendemonstrated to be formed as a result of cleavage in the vicinity of amembrane penetrating region of a membrane protein referred to as amyloidprecursor protein (APP). As a result of etiogenic gene analysis ofhereditary Alzheimer's disease, since it was found that a mutationoccurs in the APP gene itself resulting in increased production of Aβ,or production of Aβ increases due to mutation of the presenilin gene, adifferent etiogenic gene, and that Aβ extracted from the body orsynthesized artificially exhibits toxicity on nerve cells, the idea thatthe mechanism of occurrence of Alzheimer's disease involves excessivelyproduced Aβ becoming insoluble causing it to be deposited in nerve cellsand demonstrate toxicity which in turn causes degeneration is consideredto be the most promising.

In addition to Alzheimer's disease, in disorders such as Huntington'sdisease, spinal and bulbar atrophy, Machado-Joseph's disease,denatorubropallidoluysian atrophy, the accumulation and aggregation ofpolyglutamine formed due to the elongation of a CAG repeat within thegene, and in prion diseases such as Creutzfeldt-Jakob's disease, theaccumulation and aggregation of abnormal protein caused by structuralconversion of normal prion protein by some unknown cause, have beendetermined to be the cause of neuropathy/cell death in each of thesediseases. Moreover, in Parkinson's disease and Lewy body disease, theaccumulation and deposition of a protein known as α-cynucrein, and inamyotrophic lateral sclerosis, the accumulation and aggregation of amutant superoxide dismutase, have been indicated has having thepotential to cause neuropathy/cell death. In addition, among these,although prion protein and α-cynucrein adopt a β sheet structure in thesame manner as Aβ, this has been determined to function as the triggerthat causes aggregation and deposition.

Thus, if it were possible to produce a model that expresses apathological state similar to that of human disease by making abnormalproteins thought to cause these neurodegenerative diseases present inexcess in the body of an animal, that model could be considered to beextremely useful in terms of developing etiogenic therapy forneurodegenerative diseases.

Attempts have previously been made to produce an animal model ofAlzheimer's disease either by producing Aβ in excess in an animal bodyby transgenic mouse technology, or by inducing a disorder by directlyinjecting Aβ into the brain of a normal animal. For example, decreasedlearning and memory ability has been reported by implanting aminiaturized osmotic pressure pump beneath the skin of the back of anormal rat for the purpose of continuous infusion of β protein into theventricle, (Neuroscience Letters, Vol. 170, pp. 63-66, 1994). This βprotein ventricular infusion model is the most suitable as a system forevaluating Alzheimer's disease therapeutic agents used for the purposeof etiogenic therapy.

On the other hand, prostaglandin (PG) compounds are known to havevarious physiological activities, including potent platelet aggregationinhibitory action, vasodilation and its accompanying blood pressurelowering action, gastric acid secretion inhibitory action, smooth musclecontractile action, cell protective action and diuretic action. Numerousattempts have been made to develop natural PG present in the body, or PGderivatives synthesized in the form of their agonists, aspharmaceuticals based on these physiological activities, and some ofthose attempts have lead to pharmaceuticals that have actually beenmarketed commercially.

Among PG, natural prostacyclins are locally acting hormones producedprimarily in the vascular endothelium in the body, and attempts havebeen made to use them directly as pharmaceuticals by utilizing theirpotent physiological activity such as platelet aggregation inhibitoryaction and vasodilatory action (P. J. Lewis, J. O. Grady, ClinicalPharmacology of Prostaglandin). However, since natural prostacyclinshave an enol-ether bond within their molecules that is susceptible tohydrolysis, they have the problem of being easily deactivated underneutral or acidic conditions, thereby preventing them from beingpreferable compounds for use as pharmaceuticals due to their chemicalinstability. Thus, research has been conducted on the synthesis ofchemically stable synthetic prostacyclin derivatives that exhibitsimilar activity to that of natural prostaglandins (Synthesis, 1984,449, Japanese Unexamined Patent Publication No. 61-129146).9(O)-methano-Δ^(6(9α))-prostaglandin I₁ (isocarbocyclines) has beensynthesized that adequately satisfies chemical stability by substitutingmethine groups (—CH═) for the oxygen atoms at the 6^(th) and 9^(th)positions of prostacycline (Japanese Unexamined Patent Publication No.59-210044), and this compound has demonstrated potent plateletaggregation inhibitory action, vasodilatory blood pressure loweringaction and other biological activities comparable to naturalprostaglandins (Japanese Unexamined Patent Publication No. 59-210044,Japanese Unexamined Patent Publication No. 61-197518).

In the past however, development of PGs as pharmaceuticals has primarilytaken place in the obstetrics and gynecology, cardiovascular andgastrointestinal fields. In addition, they have also been indicated asbeing useful as oral therapeutic agents for diabetes (JapaneseUnexamined Patent Publication No. 2-167227). However, PG compounds alsohave the potential for being useful as pharmaceuticals in the field ofneurology and psychiatry.

Namely, PGD₂, PGE₁ or the isocarbacycline derivative mentioned above hasbeen shown to demonstrate cerebral protective action on animals in ahypoxic state (Japanese Unexamined Patent Publication No. 60-146826,Japanese Unexamined Patent Publication No. 4-187637, Brain Research,Vol. 769, pp. 321-328, 1997).

In addition, it has also been reported that PGD₂, PGE₁, PGE₂ or PGF_(2α)has a process extension promoting action on neuroblastoma cells(Bulletin of the Japanese Society for Neurochemistry, Vol. 24, 376,1985; Japanese Pharmacology and Therapeutics, Vol. 21, 37, 1993), thatPGI₂ and PGE₂ have a protective action on primary cultured nerve cells(Neuroscience Letters, Vol. 160, 106, 1993); Brain Research, Vol. 663,237, 1994), and that PGD₂, PGJ₂ and so forth have an action thatpromotes production of nerve growth factor (Japanese Unexamined PatentPublication No. 7-291867).

However, none of these reports specifically indicate the potential forPGs being able to be used as therapeutic agents for neurodegenerativediseases.

However, in the case of attempting to develop a pharmaceutical in thefield of neurology and psychiatry, there are problems resulting from thediverse actions possessed by PGs as described above causing adverse sideeffects, and in order to solve these problems, it is necessary to obtaina compound that acts as specifically as possible on the brain andnervous system. In addition, another problem is the vascular system ofthe brain restricting the permeability of certain compounds due to thepresence of the so-called blood-brain barrier, and in order to develop aPG as a pharmaceutical, it is necessary to enhance the permeability ofthat PG through the blood-brain barrier.

Therefore, as a result of conducting an in vitro autoradiographicevaluation in a large coronal section of the cerebral hemisphere ofJapanese monkeys using a labeled prostacyclin derivative([³H]iloprost-Schering), the inventors of the present invention foundprostacyclin bonding sites in the striatum, amygdala nucleous,hippocampus and a portion of the cerebral cortex. In addition, the[³H]iloprost binding sites found here differed from the binding sites of[³H]PGE₂, and PGE₂ and PGE₁ were determined to recognize the samereceptors. In platelets, iloprost binding sites also react with PGE₁,and are known to be completely different from PGE₂ receptors.

During the course of the above research, a novel PGI₂ receptor has beendetermined to exist in the central nervous system (Neuroscience, Vol.65, pp. 493-503, 1995), and certain of the inventors of the presentinvention found several types of isocarbacycline derivatives thatfunction as specific ligands of this novel PGI₂ receptor present in thecentral nervous system (Japanese Unexamined Patent Publication No.8-245498, Japanese Unexamined Patent Publication No. 10-87608, JapaneseUnexamined Patent Publication No. 10-10610, Japanese Unexamined PatentPublication No. 11-5764 and Journal of Neurochemistry, Vol. 72, pp.2583-2592, 1999). These isocarbacycline derivatives have demonstratedprotective action on cultured nerve cells and animal cerebral nervecells in a hypoxic state (EP-911314).

On the other hand, it has been reported that stability can be improvedby formulating PGE₁, PGA₁ or the above isocarbacycline derivative as alipid microshere preparation (Japanese Unexamined Patent Publication No.58-222014, Japanese Unexamined Patent Publication No. 59-141518 andJapanese Unexamined Patent Publication No. 61-289034). Moreover,penetration to the brain when administered into the blood has been shownto increase by formulating the methyl ester of the isocarbacyclinederivative as a lipid emulsion (J. Pharm. Pharmacol., Vol. 48, pp.1016-1022, 1996).

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a clinicallyapplicable neurodegenerative disease therapeutic agent, a therapeuticagent for degenerative diseases that present dementia symptoms and,particularly, an Alzheimer's disease therapeutic agent having highlearning and memory disorder improvement action and minimal adverse sideeffects such as toxicity and blood pressure lowering action for use as aneurodegenerative disease therapeutic agent, therapeutic agent fordegenerative diseases that present dementia symptoms, and particularlyan Alzheimer's disease therapeutic agent.

As a result of repeatedly conducting earnest research based on the aboveproblems, the inventors of the present invention first found that byusing an evaluation system an animal model of Alzheimer's disease bycontinuous intraventricular β-amyloid infusion, specific isocarbacyclinederivatives that are specific ligands of a novel PGI₂ receptor presentin the central nervous system have the action of improving learning andmemory disorders caused by β-amyloid protein, and that these compoundshave hardly any effect on the peripheral cardiovascular system, and thattheir action is highly brain-specific, thereby leading to completion ofthe present invention.

Namely, the present invention is a neurodegenerative disease therapeuticagent containing as its active ingredient a (15R)-isocarbacyclinederivative indicated with the following formula [I]:

(wherein, R₁ represents a C₁-C₆ alkylene group, and R₂ represents ahydrogen atom, a C₁-C₇ alkyl group or protective group),

-   -   a neurodegenerative disease therapeutic agent containing as its        active ingredient a 15-deoxy-isocarbacycline derivative        indicated with the following formula [III]:        (wherein, R₁ and R₂ are the same as defined in formula [I]),    -   a neurodegenerative disease therapeutic agent in which the above        neurodegenerative disease is a degenerative disease that        presents dementia symptoms, and a neurodegenerative disease        therapeutic agent in which said neurodegenerative disease is        Alzheimer's disease.

BEST MODE FOR CARRYING OUT THE INVENTION

In the above formulas [I] and [III], R₁ is a C₁-C₆ alkylene group, andmore specifically, a linear or branched alkylene group such as thatrepresented with —(CH₂)_(n)— (wherein, n represents an integer of 1 to6), and n is preferably 1 to 4, and particularly preferably 1. In theabove formulas [I]and [III], although the substitution position of themethyl group on the tolyl group on the omega chain may be the orthoposition, meta position or para position, the meta position ispreferable.

R₂ represents a hydrogen atom, a C₁-C₇ alkyl group or a protectivegroup. Specific examples of a C₁-C₇ alkyl group include linear orbranched alkyl groups such as a methyl group, an ethyl group, ann-propyl group, an iso-propyl group, an n-butyl group, a sec-butylgroup, a tert-butyl group or an n-pentyl group.

Examples of R₂ protective groups are represented by a pharmaceuticallyacceptable salt or ester. Specific examples of salts include, as acidaddition salt, mineral acid salts such as chloride, bromide, iodide,phosphate, nitrate and sulfate, organic sulfonates such asmethanesulfonate, 2-hydroxyethanesulfonate and p-foluenesulfonate,organic carboxylates such as acetate, trifluoroacetate, propionate,oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate,malate or mandelate, and as salt of base, organic sulfonates such asmethanesulfonate, 2-hydroxyethanesulfonate and p-toluenesulfonate, saltsof inorganic bases such as sodium salt, potassium salt, magnesium salt,calcium salt and aluminum salt, and salts of organic bases such asmethylamine salt, ethylamine salt, lysine salt and ornithine salt. Inaddition, examples of esters include C₁-C₅ alkyl esters, specificexamples of which include methyl ester and ethyl ester.

Preferable examples of the compound in the above formula [I] include(15R)-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline represented withthe following formula [III] and its methyl ester form.

In addition, preferable examples of the compound in the above formula[III] include 15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclinerepresented with the following formula [IV] and its methyl ester form.

The compounds of these formulas [I] through [IV] can be producedaccording to the method disclosed in Japanese Unexamined PatentPublication No. 8-245498 or Japanese Unexamined Patent Publication No.11-5764 previously filed by certain of the inventors of the presentinvention.

Although there are no particular restrictions on the application targetof the neurodegenerative disease therapeutic agent of the presentinvention, it is particularly useful for mammals, and can be usedespecially preferably for livestock, laboratory animals, pets andhumans. Although there are no particular restrictions on the targetdiseases provided it is a disease that is caused by neurodegeneration,it is specifically effective in application to degenerative diseasesthat present dementia symptoms such as Alzheimer's disease and Pick'sdisease, and application to Alzheimer's disease is particularlyeffective.

Although there are no particular restrictions on the administrationmethod, preferable examples of administration methods include oraladministration, percutaneous administration, nasal administration,intravenous administration, intraperitoneal administration, intrarectaladministration and intraventricular administration. When clinicallyapplying the isocarbacycline derivative used in the present invention orits clathrate compound, the isocarbacycline derivative as the activeingredient is preferably prepared in the form of a pharmaceuticalcomposition comprised of a pharmaceutically acceptable carrier such as asolid or liquid, followed by the addition of a diluent, namely anadditive such as a vehicle or stabilizer, as necessary. An injectableadministration preparation of the isocarbacycline derivative of thepresent invention to be used for therapeutic administration mustnormally be in a sterile state. Sterility is achieved easily byfiltering through a sterilization filtration membrane such as a membranefilter having a pore size of 0.2 μm.

In the above pharmaceutical composition, the ratio of the above activeingredient to the carrier component can be varied between, for example,0.000001-90% w/w. Although dependent upon the administration method,age, target disease and so forth, the therapeutically effective dosagecan be 0.01 μg-1000 mg/day/person, and preferably 0.01 μg-10mg/day/person. The absorption efficiency into the body is preferablydetermined individually for each compound according to well knownpharmacological methods with respect to each administration route.

Examples of dosage forms and administration forms include oraladministration using a dosage form such as granules, grains, powders,pills, tablets, capsules or liquids, and parenteral administration usinga local preparation such as suppositories, aerosols, ointments and skinpatches. Administration may also be performed by intravenousadministration, intraarterial administration, intramuscularadministration and subcutaneous administration using an injectablepreparation. In addition, an injectable powder may also be used bypreparing at the time of use. Moreover, administration may also beperformed by nasal administration, intraperitoneal administration,intrarectal administration or intraventricular administration.

Pharmaceutical organic or inorganic and solid or liquid carriers ordiluents suitable for oral, enteric or parenteral administration can beused for preparing the isocarbacycline derivative as claimed in thepresent invention in the form of a pharmaceutical preparation.

Examples of typical carriers or diluents that can be incorporated intablets, capsules and so forth include binders such as acacia,cornstarch and gelatin, vehicles such as microcrystalline cellulose,disintegration agents such as cornstarch and alginic acid, lubricantssuch as magnesium stearate, and sweeteners such as sucrose and lactose.In the case the dosage form is a capsule, a liquid carrier such as fattyoil may be contained in addition to the above substances. Various typesof other substances can be used as coating agents or agents forimproving physical properties in dosage units. Sterile compositions forinjection can be formulated in accordance with conventionalpharmacological methods. For example, it is preferable to dissolve orsuspend the active compound in a vehicle such as water or naturalvegetable oil or a synthetic fat vehicle such as ethyl oleate. Bufferssuch as citrate, acetate and phosphate buffers as well as antioxidantssuch as ascorbic acid can also be incorporated in accordance withallowed pharmaceutical methods.

In preparing in the form of tablets, tablets can be formed in accordancewith routine methods using a vehicle such as lactose, starch orcrystalline cellulose, a binder such as carboxymethyl cellulose, methylcellulose or polyvinyl pyrrolidone, and a disintegration agent such assodium alginate, sodium bicarbonate or sodium lauryl sulfate.

Pills, powders and granules can be similarly formed in accordance withroutine methods using the above vehicles and so forth. Liquids andsuspensions can be formed in accordance with routine methods usingglycerin esters such as tricaprilin and triacetin or alcohols such asethanol. Capsules are formed by filling granules, powders or liquidsinto gelatin or other capsules.

In the case of preparations for oral administration, the isocarbacyclinederivative as claimed in the present invention can be converted to acyclodextrin clathrate compound. Clathrate compounds are prepared byadding a solution in which cyclodextrin has been dissolved in waterand/or an organic solvent that mixes easily with water to a solution inwhich isocarbacycline has been dissolved in an organic solvent thatmixes easily with water. The target cyclodextrin clathrate compound isthen isolated by heating the mixture followed by concentrating underreduced pressure, filtering while cooling or separating the product bydecantation. The ratio of organic solvent and water varies according tothe solubility of the starting materials and product. It is preferablethat the temperature within the cyclodextrin clathrate compoundpreparation does not exceed 70° C. α-, β- and γ-cyclodextrin or mixturesthereof can be used to prepare a cyclodextrin clathrate compound. Thestability of isocarbacyclines can be improved by converting to acyclodextrin clathrate compound.

Examples of dosage forms for subcutaneous, intramuscular or intravenousadministration include injectable preparations in the form of an aqueousor non-aqueous solution. Physiological saline, for example, is used foraqueous solutions. Propylene glycol, polyethylene glycol, olive oil,ethyl oleate and so forth are used for non-aqueous solutions, andantiseptics, stabilizers and so forth are added to these as necessary.Injectable preparations are sterilized by suitably performing proceduressuch as filtering through a bacteria capturing filter or blending in adisinfectant and so forth.

Examples of dosage forms for percutaneous administration includeointments and creams. Ointments are formed in accordance with routinemethods using oils such as castor oil and olive oil or Vaseline, whilecreams are formed in accordance with routine methods using emulsifierssuch as fatty oil, diethylene glycol and sorbitan monofatty acid ester.

Ordinary suppositories such as soft gelatin capsules are used for rectaladministration.

Preparations for parenteral administration can also be administered asan emulsion. Namely, fat emulsions prepared by adding water to a uniformsolution of vegetable oil such as soy bean oil, phospholipid such aslecithin and isocarbacycline as claimed in the present inventionfollowed by homogenizing with a homogenizer such as a pressure sprayinghomogenizer or ultrasonic homogenizer, can also be used as injectablepreparations.

Although the following provides a more detailed explanation of thepresent invention through examples, the present invention is not limitedin any way to these examples.

EXAMPLES

Test compounds A through C and comparative test compound D used in thefollowing examples are the compounds indicated below.

Test Compound A:

-   (15R)-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline

Test Compound B:

-   (15R)-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline methyl ester

Test Compound C:

-   15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline methyl    ester

Comparative Test Compound D:

-   (15S)-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline methyl ester

Reference Example 1 Measurement Method of Learning and Memory Ability ofRats by a Step-Through Passive Evasion Test

A step-through passive evasive reaction apparatus composed of twochambers separated by a guillotine door, was used for the experimentalapparatus. Namely, one of the chambers was a bright chamber composed ofclear acrylic boards (floor: 15 cm×25 cm, height: 15 cm) while the otherchamber was a dark chamber (of the same size) composed of black acrylicboards. In addition, stainless steel grids having a diameter of 4 mmwere provided at intervals of 15 mm on the floor of the dark room, andconnected to a shock generator for applying an electric shock.

To begin with, after opening the guillotine door and allowing to freelyexplore the inside of the apparatus for 1 minute, the door was closed,the rat was placed in the bright chamber as an acquisition trial and thedoor was opened 30 seconds later. The door was closed after all fourlimbs of the rat entered the dark chamber followed immediately by theapplication of an electric shock. The intensity of the electric shockwas set at 0.5 mA for 5 seconds. Subsequently, the rat was immediatelyplaced in the bright chamber and training was repeated while followingthe same procedure until the rat remained in the bright chamber for 120seconds even if the guillotine door was opened. As a retention trialconducted 24 hours after the acquisition trial, the rat was placed inthe bright chamber and the amount of time until all four limbs enteredthe dark chamber after the guillotine door was opened 30 seconds laterwas measured (step-through latency). The maximum observation time duringthe retention trial was set at 300 seconds.

Reference Example 2 Production of a Alzheimer's Dementia Animal Model byContinuous Intraventricular Infusion of β Protein

Seven-week-old, male Wistar rats (body weights: 220-250 g) were used(N=5-10).

β-amyloid protein (1-40) or β-amyloid protein (1-42) was dissolved in35% acetonitrile/0.1% TFA, injected into a mini-osmotic pressure pump(volume: 230 μl, 0.5 μl/hour) at the rate of 300 pmol/day, and thenconnected with a dental injection needle by means of a polyethylenetube. For the control group, a pump was connected and injected withβ-amyloid protein (40-1). After anesthetizing the rats withpentobarbital (50 mg/kg, i.p.), an incision was made in the skin on thehead and a hole was drilled in the cranium with a microdrill inaccordance with the brain map. The injection needle was inserted sp thatthe tip of the needle entered the later ventricle (A=−0.3 mm, L=1.2 mm,H=4.5 mm), and immobilized with dental cement. An osmotic pressure pumpwas embedded beneath the skin on the back.

Taking the day on which the procedure for embedding the mini-osmoticpump beneath the skin to be day 0, passive evasion tests were conductedon days 13 and 14 in accordance with the method indicated in ReferenceExample 1. As a result, learning and memory ability was confirmed tohave decreased in the β-amyloid protein (1-40) or β-amyloid protein(1-42) dose group as compared with the β-amyloid protein (40-1) dosegroup.

Reference Example 3 Toxicity Study Method

Thirty-five six-week-old, male C57BL mice were divided into 7 groups asshown in Table 1 below.

TABLE 1 Dosing Dosing solution solution Administered Dosage volumeconcentration No. of substance mg/kg ml/kg mg/ml animals Solvent only 05 0 5 Test compound B 0.03 5 0.006 5 Test compound B 0.3 5 0.06 5 Testcompound B 3 5 0.6 5 Test compound C 0.03 5 0.006 5 Test compound C 0.35 0.06 5 Test compound C 3 5 0.6 5

The test compound solutions were administered into the caudal vein atthe rate of about 5 ml per minute. The general condition of each animalwas suitably observed immediately before and immediately after dosing onthe day of dosing, and at 15 minutes, 30 minutes, 60 minutes, 2 hours, 4hours and 6 hours after dosing. Moreover, the animals were observed forgeneral condition once in a day in the morning from the day after dosingthrough day 14. In addition, body weights were measured immediatelybefore dosing and on days 1, 3, 7 and 14 after dosing.

Reference Example 4 Measurement Method of Blood Pressure Lowering Action

Thirty five, male Wistar rats. (body weights: 230-270 g) were dividedinto 7 groups as shown in Table 2 below.

TABLE 2 Dosing Dosing solution solution Administered Dosage volumeconcentration No. of substance mg/kg ml/kg mg/ml animals Solvent only 05 0 5 Test compound B 0.03 5 0.006 5 Test compound B 0.3 5 0.06 5 Testcompound B 3 5 0.6 5 Comp. test compound D 0.03 5 0.006 5 Comp. testcompound D 0.3 5 0.06 5 Comp. test compound D 3 5 0.6 5

Cannulas were inserted into the left carotid artery for measuring bloodpressure, and into the left jugular vein for intravenous injection ofthe test compounds, respectively. After being housed normally in theircages overnight following surgery, the animals were injected with thetest solutions without anesthesia. Blood pressure and heart rate weremeasured immediately before dosing (0 minutes) and at 5, 30, 60, 120 and240 minutes after dosing. The blood pressure at 0 minutes for eachanimals was assigned a value of 100, and measured values of bloodpressure at each time were then normalized based on that value.

Example 1 Measurement of Learning and Memory Ability Improvement Effect

The learning and memory ability improvement action of test compound Awas measured using the evaluation methods of Reference Examples 1 and 2.Test compound A was dissolved in phosphate-buffered physiological salineand injected using an osmotic pressure pump in the same manner asinjection of β-amyloid protein. Test compound A was dissolved inβ-amyloid protein solution and injected simultaneous to β-amyloidprotein using an osmotic pressure pump.

TABLE 3 Time until moved to dark chamber during retention trial Mean ±standard error (units: sec.) β-amyloid protein (40-1) 300 pmol/day 272.1± 18.3 (n = 8)  dose group β-amyloid protein (1-42) 300 pmol/day 192.5 ±27.4 (n = 11) dose group β-amyloid protein (1-42) 300 pmol/day + 265.7 ±32.6 (n = 6)  test compound A 1.2 fmol/day dose group β-amyloid protein(1-42) 300 pmol/day + 261.8 ± 26.7 (n = 11) test compound A 12 fmol/daydose group β-amyloid protein (1-42) 300 pmol/day + 276.1 ± 17.0 (n = 10)test compound A 120 fmol/day dose group

In other words, in this study, test compound A exhibited action thatimproved learning and memory ability. In particular, in those animals ofthe test compound 12 fmol/day dose group and 120 fmol/day dose group,the amount of time until the animals moved into the dark chamberincreased significantly as compared with the group dosed with β-amyloidprotein (1-42) only (p>0.05).

Example 2 Toxicity Study

Ace A toxicity study was conducted on test compounds B and C. As aresult, none of the animals died in any of the groups. In the case oftest compound B, although decreased movement was observed in the 3 mg/kgdose group starting immediately after dosing, the change was extremelymild and disappeared by 30 minutes after dosing. In the case of testcompound C, there were no abnormalities observed in any of the groups.Moreover, there were no abnormalities observed in any of the groups forboth test compounds B and C starting on the day after dosing. Inaddition, there were also no significant fluctuations in body weightsfor test compound B or C.

In other words, both test compounds were clearly determined to haveextremely low toxicity.

Example 3 Measurement of Blood Pressure Lowering Action

Fluctuations in blood pressure following administration of test compoundB and comparative test compound D were as shown in Table 4 below.

TABLE 4 Blood pressure (mmHg, ± standard error) Admin. Dosage 0 5 30 60substance mg/kg min. min. min. min. 2 hr. 4 hr. Solvent 0 100 104.9103.9 107.5 108.5 106.7 only (3.0) (2.3) (1.3) (2.0) (2.5) Test 0.03 100105.8 103.4 103.6 104.5 100.5 comp. B (2.7) (3.0) (2.9) (3.7) (4.7)Test. 0.3 100 92.9 97.7 97.7 98.5 98.0 Comp. B (2.2) (2.3) (2.7) (3.0)(5.0) Test 3 100 80.4 99.4 101.9 102.7 101.0 comp. B (3.9) (3.6) (2.2)(2.3) (2.5) Comp. 0.03 100 88.9 92.1 96.5 97.3 99.0 test (7.8) (2.6)(3.0) (1.8) (3.3) comp. D Comp. 0.3 100 49.6 78.5 94.4 93.3 99.3 test(2.0) (3.8) (2.0) (2.2) (3.0) comp. D Comp. 3 100 55.0 60.0 81.1 103.8115.4 test (1.2) (1.9) (4.3) (3.4) (3.7) comp. D

In addition, the fluctuations in heart rate following administration oftest compound B and comparative test compound D were as shown in Table5.

TABLE 5 Heart rate (standard error) Admin. Dosage 0 5 30 60 substancemg/kg min. min. min. min. 2 hr. 4 hr. Solvent 0 100 111.2 111.5 110.4111.4 111.2 only (3.4) (5.5) (2.4) (3.5) (2.6) Test 0.03 100 114.8 123.8110.9 103.8 100.5 comp. B (6.7) (6.4) (3.4) (2.2) (4.0) Test. 0.3 100112.2 108.4 101.6 104.7 112.0 Comp. B (4.2) (2.9) (5.2) (5.5) (5.7) Test3 100 135.0 122.7 102.5 101.4 102.2 comp. B (4.8) (4.6) (4.1) (5.8)(6.4) Comp. 0.03 100 135.5 113.7 106.1 102.9 104.3 test (4.3) (1.8)(2.5) (3.4) (2.1) comp. D Comp. 0.3 100 127.0 109.1 96.3 97.0 94.4 test(2.0) (1.7) (3.6) (3.0) (2.5) comp. D Comp. 3 100 124.9 142.7 131.0104.7 104.1 test (8.1) (5.2) (6.7) (6.5) (5.1) comp. D

Although test compound B caused a decrease in blood pressure and anincrease in heart rate immediately after dosing in the 3 mg/kg dosegroup, the blood pressure and the heart rate recovered rapidly, theblood pressure, in particular, only demonstrating a mild decrease at 5minutes after dosing, and both parameters were observed to return tonormal at 30 minutes after dosing. There were no significantfluctuations observed in the 0.3 and 0.03 mg/kg dose groups.

On the other hand, in the case of comparative test compound D, decreasedblood pressure was observed to continue for 30 minutes or more in the 3mg/kg and 0.3 mg/kg dose groups.

Namely, test compound B was clearly determined to have an extremely mildeffect on the circulatory system.

INDUSTRIAL APPLICABILITY

The therapeutic agent containing as active ingredient a specificisocarbacycline derivative of the present invention is a clinicallyapplicable neurodegenerative disease therapeutic agent, therapeuticagent for degenerative diseases that present dementia symptoms and, inparticular, an Alzheimer's disease therapeutic agent, that is highlyeffective in improving learning and memory disorders and has minimaladverse side effects such as toxicity and blood pressure loweringeffects, which can be used as a neurodegenerative disease therapeuticagent, therapeutic agent for degenerative diseases that present dementiasymptoms, and in particular an Alzheimer's disease therapeutic agent.

1. A method of treating neurode generative disease, said methodcomprising administering to a subject in need of treatment an effectiveamount of a (15R)-isocarbacycline compound represented by the followingformula [I]:

wherein, R₁ represents a C₁-C₆ alkylene group, and R₂ represents ahydrogen atom, a C₁-C₇ alkyl group or a protective group.
 2. A method oftreating neurodegenerative disease according to claim 1, said methodcomprising administering to a subject in need of treatment an effectiveamount of a (15R)-16-m-tolyl)-17,18,19,20-tetranorisocarbacyclinerepresented by the following formula [II] or its methyl ester form:


3. A method of treating a neurodegenerative disease according to claim 1or 2, wherein said neurodegenerative disease is a degenerative diseasethat presents dementia symptoms.
 4. A method of ting a neurodegenerativedisease according to claim 1 or 2, wherein said neurodegenerativedisease is Alzheimer's disease.
 5. A method of treatingneurodegenerative disease, said method comprising administering to asubject in need of treatment a 15-deoxy-isocarbacycline compoundrepresented by the following formula [III]:

wherein, R₁ represents a C₁-C₆ alkylene group, and R₂ represents ahydrogen atom, a C₁-C₇ alkyl group or a protective group.
 6. A method oftreating a neurodegenerative disease according to claim 5, said methodcomprising administering to a subject in need of treatment a15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacycline represented bythe following formula [IV] or its methyl ester form:


7. A method of treating a neurodegenerative disease according to claim 5or 6, wherein said neurodgenerative disease is a degenerative diseasethat presents dementia symptoms.
 8. A method of treating aneurodegenerative disease according to claim 5 or 6, wherein saidneurodegenerative disease is Alzheimer's disease.